scholarly journals An additional acidic residue in the membrane portion of the b-subunit of the energy-transducing adenosine triphosphatase of Escherichia coli affects both assembly and function

1984 ◽  
Vol 221 (1) ◽  
pp. 43-51 ◽  
Author(s):  
D A Jans ◽  
A L Fimmel ◽  
L Hatch ◽  
F Gibson ◽  
G B Cox
Keyword(s):  
Escherichia Coli ◽  
Oxidative Phosphorylation ◽  
Atpase Activity ◽  
Dna Sequence ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
The Other ◽  
Multicopy Plasmid ◽  
B Subunit ◽  
And Function

Glycine at position 9 is replaced by aspartic acid in the mutant b-subunit of Escherichia coli F1F0-ATPase coded for by the uncF476 allele. The mutant b-subunit is not assembled into the membrane in haploid strains carrying the uncF476 allele, but, if the mutant allele is incorporated into a multicopy plasmid, then some assembly of the mutant b-subunit occurs. Two revertant strains were characterized, one of which (AN2030) was a full revertant, the other (AN1953) a partial revertant. DNA sequencing indicated that in strain AN2030 the uncF476 mutation had reverted to give the sequence found in the normal uncF gene. The partial-revertant strain AN1953, however, retained the DNA sequence of the uncF476 allele, and complementation analysis indicated that the second mutation may be in the uncA gene. Membranes prepared from the partial-revertant strain carried out oxidative phosphorylation, although the membranes appeared to be impermeable to protons, and the ATPase activity was sensitive to the inhibitor dicyclohexylcarbodi-imide.

scholarly journals Genetic complementation between two mutant unc alleles (unc A401 and unc D409) affecting the F1 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12

1978 ◽  
Vol 170 (3) ◽  
pp. 593-598 ◽  
Author(s):  
G B Cox ◽  
J A Downie ◽  
F Gibson ◽  
J Radik
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Fluorescence Quenching ◽  
Oxidative Phosphorylation ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
The Other ◽  
Genetic Complementation ◽  
Magnesium Ion ◽  
Adenosine Triphosphatase Activity

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. A genetic-complementation analysis, using partial diploid strains, showed that the new mutant allele, uncD409, is in a gene distinct from the other previously identified genes uncA, uncB and uncC. A strain carrying the uncd409 allele has no Mg2+ ion-stimulated adenosine triphosphatase activity and is therefore phenotypically similar to strains carrying the uncA401 mutant allele. Complementation between the uncA401 and the uncD409 alleles occurred, as indicated by growth of partial diploid strains on succinate and their growth yields on limiting concentrations of glucose. Complementation was confirmed by using membranes prepared from the above partial diploids. Such membranes were found to have Mg2+-stimulated adenosine triphosphatase activity, ATP-dependent transhydrogenase activity ADP-induced atebrin-fluorescence quenching and low but significant amounts of oxidative phosphorylation.

scholarly journals Partial diploids of Escherichia coli carrying normal and mutant alleles affecting oxidative phosphorylation

1977 ◽  
Vol 162 (3) ◽  
pp. 665-670 ◽  
Author(s):  
F Gibson ◽  
G B Cox ◽  
J A Downie ◽  
J Radik
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Quenching ◽  
Oxidative Phosphorylation ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Growth Yields ◽  
Normal Allele ◽  
Adenosine Triphosphatase Activity

A plasmid was isolated which included the region of the Escherichia coli chromosome carrying the known genes concerned with oxidative phosphorylation (unc genes). This plasmid was used to prepare partial diploids carrying normal unc alleles on the episome and one of the three mutant alleles (unc A401, uncB402 or unc-405) on the chromosome. These strains were compared with segregants from which the plasmid had been lost. Dominance of either normal ormutant unc alleles was determined by growth on succinate, growth yields on glucose, Mg-ATPase (Mg2+-stimulated adenosine triphosphatase) activity, atebrin-fluorescence quenching, ATP-dependent transhydrogenase activity and oxidative phosphorylation. In all the above tests, dominance of the normal allele was observed. However, in membranes from the diploid strains which carried a normal allele and either of the mutant alleles affecting Mg-ATPase activity (uncA401 or unc-405), the energy-linked functions were only partially restored.

The Leeuwenhoek Lecture, 1981 - The biochemical and genetic approach to the study of bioenergetics with the use of Escherichia coli : progress and prospects

1982 ◽  
Vol 215 (1198) ◽  
pp. 1-18 ◽  
Keyword(s):  
Escherichia Coli ◽  
Cell Membrane ◽  
Oxidative Phosphorylation ◽  
Adenosine Triphosphatase ◽  
Atp Synthesis ◽  
Genetic Approach ◽  
Biochemical Genetics ◽  
Multiprotein Complex ◽  
E Coli ◽  
And Function

How the ‘energy currency’ of the cell, adenosine triphosphate (ATP), is produced consequent upon the oxidation of foodstuffs (oxidative phosphorylation) is, despite prolonged research, still a matter of debate and the molecular mechanism of the process is unknown. It appears that the problem of oxidative phosphorylation can be approached with the aid of the biochemical genetics of the bacterium Escherichia coli . The ease of manipulation of bacteria and definitive results obtained by this approach have been invaluable in solving other major biochemical problems. Mutants affected in oxidative phosphorylation have been isolated and characterized by genetic and biochemical techniques. These ‘ unc ' mutants are affected in the adenosine triphosphatase (ATPase) multiprotein complex which is part of the cell membrane and responsible for the terminal stages of ATP synthesis. Seven distinct genes concerned with oxidative phosphorylation have been characterized in E. coli and shown to be part of an operon. The relationships between the different classes of unc genes and the various components of the ATPase have been established. Information about the assembly of the ATP synthesizing complex in the cell membrane has also been obtained and the stage set for further studies on the assembly, control and function of the ATP synthesizing system.

scholarly journals Properties of membranes from mutant strains of Escherichia coli in which the β-subunit of the adenosine triphosphatase is abnormal

1979 ◽  
Vol 180 (1) ◽  
pp. 111-118 ◽  
Author(s):  
A E Senior ◽  
D R Fayle ◽  
J A Downie ◽  
F Gibson ◽  
G B Cox
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
Beta Subunit ◽  
Beta Subunits ◽  
F1 Atpase ◽  
Mutant Strains ◽  
Membrane Bound ◽  
Phenotypic Variations

Five uncoupled mutant strains of Escherichia coli carrying mutations in the uncD gene have been studied. In each of these mutant strains the beta-subunit of the F1 portion of the membrane-bound adenosine triphosphatase is abnormal. In one of the mutant strains (carrying the uncD12 allele) in F1-ATPase aggregate was formed which was purified and found to have low ATPase activity. ATPase activity was absent in the other four strains and the abnormal beta-subunits were tightly bound to the membranes. However, membranes from these strains exhibited various proton permeabilities as indicated by NADH-dependent atebrin-fluorescence quenching and bound different amounts of normal F1-ATPase. The amounts of reconstitution of energy-linked reactions after the addition of normal F1-ATPase also varied depending on the mutant allele. It is apparent that considerable phenotypic variations can occur between strains carrying mutations in the same unc gene.

scholarly journals A mutation affecting a second component of the F0 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12. The uncC424 allele

1977 ◽  
Vol 164 (1) ◽  
pp. 193-198 ◽  
Author(s):  
F Gibson ◽  
G B Cox ◽  
J A Downie ◽  
J Radik
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Fluorescence Quenching ◽  
Mutant Strain ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
Magnesium Ion ◽  
New Mutation ◽  
Similar Phenotype

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. The new mutant strain has a similar phenotype to the uncB mutant described previously; results from reconstitution experiments in vitro indicate that the new mutation also affects a component of the F0 portion of the Mg2+-stimulated adenosine triphosphatase. A method was developed to incorporate mutant unc alleles into plasmids. Partial diploid strains were prepared in which the uncB402 allele was incorporated into the plasmid and the new unc mutation into the chromosome, or vice versa. Complementation between the mutant unc alleles was indicated by growth on succinate, growth yields on glucose, ATP-dependent transhydrogenase activities, ATP-induced atebrin-fluorescence quenching and oxidative-phosphorylation measurements. The gene in which the new mutation occurs is therefore distinct from the uncB gene, and the mutant allele was designated uncC424.

scholarly journals Differential Biological and Adjuvant Activities of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin Hybrids

2001 ◽  
Vol 69 (3) ◽  
pp. 1528-1535 ◽  
Author(s):  
Christal C. Bowman ◽  
John D. Clements
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Cholera Toxin ◽  
The Other ◽  
Toxin Gene ◽  
Wild Type ◽  
B Subunit ◽  
Heat Labile ◽  
A Subunit ◽  
Ifn Γ

ABSTRACT Two bacterial products that have been demonstrated to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. Although LT and CT have many features in common, they are clearly distinct molecules with biochemical and immunologic differences which make them unique. The goal of this study was to determine the basis for these biological differences by constructing and characterizing chimeric CT-LT molecules. Toxin gene fragments were subcloned to create two constructs, each expressing the enzymatically active A subunit of one toxin and the receptor binding B subunit of the other toxin. These hybrid toxins were purified, and the composition and assembly of CT A subunit (CT-A)-LT B subunit (LT-B) and LT A subunit (LT-A)-CT B subunit (CT-B) were confirmed. Hybrids were evaluated for enzymatic activity, as measured by the accumulation of cyclic AMP in Caco-2 cells, and the enterotoxicity of each toxin was assessed in a patent-mouse assay. The results demonstrated that LT-A–CT-B induces the accumulation of lower levels of cyclic AMP and has less enterotoxicity than either wild-type toxin or the other hybrid. Nonetheless, this hybrid retains adjuvant activity equivalent to or greater than that of either wild-type toxin or the other hybrid when used in conjunction with tetanus toxoid for intranasal immunization of BALB/c mice. Importantly, the ability of LT to induce a type 1 cytokine response was found to be a function of LT-A. Specifically, LT-A–CT-B was able to augment the levels of antigen-specific gamma interferon (IFN-γ) and interleukin 5 to levels comparable to those achieved with native LT, while CT-A–LT-B and native CT both produced lower levels of antigen-specific IFN-γ. Thus, these toxin hybrids possess unique biological characteristics and provide information about the basis for differences in the biological activities observed for CT and LT.

Defective Membrane Systems in Dystrophic Skeletal Muscle of the UM-X7.1 Strain of Genetically Myopathic Hamster

1975 ◽  
Vol 49 (4) ◽  
pp. 359-368
Author(s):  
N. S. Dhalla ◽  
A. Singh ◽  
S. L. Lee ◽  
M. B. Anand ◽  
A. M. Bernatsky ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Calcium Binding ◽  
Adenosine Triphosphatase ◽  
Calcium Uptake ◽  
Initial Rate ◽  
The Other ◽  
Membrane Systems ◽  
Dystrophic Muscle ◽  
Mitochondrial Calcium Uptake

1. The function of mitochondria, sarcotubular membranes (heavy microsomes), sarcolemma and myofibrils from the hind-leg skeletal muscle of about 60- and 150-day-old normal and myopathic (UM-X7.1) hamsters was examined. 2. The mitochondrial calcium uptake as well as mitochondrial phosphorylation and respiratory rates were lower in 60-day-old myopathic skeletal muscle, unlike 150-day-old myopathic animals, when pyruvate-malate and glutamate-malate were used as substrates. However, mitochondria from 150-day-old myopathic animals showed depressed glutamate-dependent respiratory and phosphorylation rates and succinate-supported initial rate of calcium uptake. 3. The microsomal calcium-uptake, but not calcium-binding, and Ca2+-stimulated adenosine triphosphatase (ATPase) activity of the 150-day-old myopathic skeletal muscle were lower than the control values. Although microsomal calcium-binding, calcium-uptake and ATPase activities of the 60-day-old myopathic muscle were not depressed significantly, the initial rate of calcium uptake was less than the control. 4. The sarcolemmal Ca2+-ATPase, but not Mg2+-ATPase or Na+ +K+-ATPase, activity was higher in 60-day-old myopathic muscle whereas the activities of all these enzymes from 150-day-old myopathic animals were higher than the control. On the other hand, the Na+ +K+-ATPase activities from 60- and 150-day-old myopathic animals were inhibited by ouabain to a lesser extent in comparison with the respective control values. 5. The myofibrillar Ca2+-ATPase and Mg2+-ATPase activities as well as inhibition of Mg2+-ATPase due to Na+ and K+ in myopathic muscle were no different from the control values. 6. The results reported here give further support to the view that different membrane systems of the dystrophic muscle are defective.

scholarly journals ADENOSINE TRIPHOSPHATASE: A NEUROHISTOCHEMICAL STUDY

1962 ◽  
Vol 10 (6) ◽  
pp. 731-740 ◽  
Author(s):  
D. NAIDOO
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Wistar Rat ◽  
The Other ◽  
Nuclear Staining ◽  
Adenosine Triphosphatase Activity ◽  
Bivalent Cations ◽  
Vascular Elements ◽  
The Brain ◽  
Cerebral Vascular

The location of adenosine triphosphatase in the brain has been studied in rapidly frozen-dried cerebral tissues of the Wistar rat. It is found that adenosine triphosphatase is an almost exclusively nuclear enzyme. Two tissue fractions of the cerebrum were separated, so that one sample was made up of vascular elements, and the other of neural elements. The two fractions were then studied for their adenosine triphosphatase activity, and compared with the histochemical findings. The two tissue fractions were found not to differ in the absence of bivalent cations. When Ca++ were added to the cerebral vascular suspension, ATPase activity was increased approximately 15 times, and only 3 times in the presence of Mg++. Conversely, the addition of Mg++ increased the ATPase activity of the neural fraction 200%; whereas, Ca++ was responsible for a 60% increase. This fact was detectable microscopically when Ca++ was found to intensify vascular nuclear staining, and Mg++ to increase the neuronal and glial nuclear staining. The results, histochemical and biochemical, are mutually confirmatory.

scholarly journals Oxidative phosphorylation in Escherichia coli K12. An uncoupled mutant with altered membrane structure

1974 ◽  
Vol 138 (2) ◽  
pp. 211-215 ◽  
Author(s):  
G. B. Cox ◽  
F. Gibson ◽  
L. McCann
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Membrane Structure ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
Mineral Salts ◽  
Normal Cells ◽  
E Coli ◽  
Escherichia Coli K12 ◽  
Membrane Bound

1. A new mutant strain (AN228) of Escherichia coli K12, unable to couple phosphorylation to electron transport, has been isolated. The mutant allele (unc-405), in strain AN228, was found to map near the uncA and uncB genes at about minute 74 on the E. coli genome. 2. A transductant strain (AN285) carrying the unc-405 allele is similar to the uncA and uncB mutants described previously in that it is unable to grow on succinate, gives a low aerobic yield on limiting concentrations of glucose, has a normal rate of electron transport, is unable to couple phosphorylation to electron transport, and lacks ATP-dependent transhydrogenase activity. 3. Strain AN285 (unc-405) is similar to an uncA mutant, but different from an uncB mutant, in that it is unable to grow anaerobically in a glucose–mineral-salts medium, and membrane preparations do not have Mg2+-stimulated adenosine triphosphatase activity. 4. Strain AN285 (unc-405) does not form an aggregate analogous to the membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate found in normal cells. In this respect it differs from strain AN249 (uncA−), which forms an inactive membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate.

scholarly journals Oxidative phosphorylation by mutant Escherichia coli membranes with impaired proton permeability

1983 ◽  
Vol 216 (1) ◽  
pp. 143-150 ◽  
Author(s):  
G B Cox ◽  
D A Jans ◽  
F Gibson ◽  
L Langman ◽  
A E Senior ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Oxidative Phosphorylation ◽  
Atpase Activity ◽  
Gene Dosage ◽  
Control Strain ◽  
Proton Permeability ◽  
F1f0 Atpase ◽  
F1 Atpase ◽  
C Subunit

The effect on the function of the Escherichia coli F1F0-ATPase of the substitution of leucine-31 by phenylalanine in the c-subunit of the enzyme was examined. The assembly of the mutant c-subunit requires an increased gene dosage [Jans, Fimmel, Langman, James, Downie, Senior, Ash, Gibson & Cox (1983) Biochem. J. 211, 717-726], and this was achieved by incorporation of the uncE408 or uncE463 alleles on to F-plasmids or multicopy plasmids. Membranes from strains carrying either the uncE463 or uncE408 alleles on F-plasmids or multicopy plasmids were capable of carrying out oxidative phosphorylation. In particular, membranes from strain AN1928 (pAN162, uncE463) gave phosphorylation rates and P/O ratios equal to or greater than those obtained for the control strain AN1460 (pAN45, unc+). However, the mutant membranes, on removal of the F1-ATPase, appeared to be proton-impermeable. The ATPase activity of the mutant membranes was also resistant to the inhibitor dicyclohexylcarbodi-imide.

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